Vitamin B12 deficiency

In healthy adults, vitamin B12 deficiency or cobalamin deficiency is uncommon, mainly because your body stores 1,000 to 2,000 times as much vitamin B12 (about 3 to 5 mg vitamin B12) as you’d typically eat in a day – total body stores can exceed 2,500 micrograms (2,500 μg), daily turnover is slow, and dietary intake of only 2.4 mcg/day (2.4 μg/day) is sufficient to maintain adequate vitamin B12 status (see Recommended Dietary Allowance [RDA]) ¹. Therefore, when there is little or no vitamin B12 in your diet, vitamin B12 stores (about 3 to 5 mg) may last for up to 5–10 years before the signs and symptoms of vitamin B12 deficiency are seen clinically ¹. In elderly individuals, vitamin B12 deficiency is more common mainly because of impaired intestinal absorption that can result in marginal to severe vitamin B12 deficiency in this population. The Recommended Dietary Allowance (RDA) for vitamin B12 is 2.4 micrograms per day (2.4 μg/day) for adolescents and adults. It is slightly higher for women who are pregnant (2.6 mcg/day) or breastfeeding (2.8 mcg/day). Currently, to maintain a healthy hematological status and serum vitamin B12 levels, average daily intakes of vitamin B12 from food of 5.94 mcg for men and 3.78 mcg for women aged 20 and older have been recommended ². For children aged 2–19 years old, mean daily intakes of vitamin B12 from food range from 3.76 mcg to 4.55 mcg ³. The original estimates of dietary folate and vitamin B12 requirements and recommended dietary allowances (RDAs) were based on the amount needed to avoid manifest deficiency disorders (megaloblastic anemia, with sub-acute combined degeneration of the cord in the case of vitamin B12 deficiency) and on levels observed in populations. However, these levels do not essentially represent necessary requirements ².

The signs and symptoms of vitamin B12 deficiency can take several years to appear ⁴, ⁵. The signs and symptoms of vitamin B12 deficiency can include the hallmark megaloblastic anemia (characterized by large, abnormally nucleated red blood cells) as well as low counts of white and red blood cells, platelets, or a combination; glossitis of the tongue (a condition in which your tongue becomes inflamed and swollen) (Figure 3); fatigue; palpitations; pale skin; dementia; weight loss; and infertility ⁶, ⁷, ⁵. Neurological changes, such as numbness and tingling in the hands and feet, can also occur ⁵. These neurological symptoms can occur without anemia, so early diagnosis and intervention is important to avoid irreversible damage ⁸. In addition, some studies have found associations between vitamin B12 deficiency or low vitamin B12 intakes and depression ⁹, ¹⁰, ¹¹. In pregnant and breastfeeding women, vitamin B12 deficiency might cause neural tube defects, developmental delays, failure to thrive, and anemia in babies ⁵.

Vitamin B12 deficiency with the classic blood and neurological signs and symptoms is uncommon ¹². However, low or marginal vitamin B12 status (200–300 pg/mL [148–221 pmol/L]) without these symptoms is much more common, at up to 40% in Western populations, especially in those with low intakes of vitamin B12-rich foods ¹², ¹³. The prevalence of vitamin B12 deficiency varies by cutoff level and biomarker used. For example, among adults aged 19 and older who participated in National Health and Nutrition Examination Survey (NHANES) between 1999 and 2004, the rate of low vitamin B12 levels in serum was 3% with a cutoff of less than 200 pg/mL (148 pmol/L) and 26% with a cutoff of less than 350 pg/mL (258 pmol/L) ¹⁴. Approximately 21% of adults older than 60 years of age had abnormal levels of at least one vitamin B12 biomarker ¹⁴.

In the United States and the United Kingdom, the prevalence of vitamin B12 deficiency is approximately 6% in persons younger than 60 years, and nearly 20% in those older than 60 years.1 Latin American countries have a clinical or subclinical deficiency rate of approximately 40% ⁴. The prevalence is 70% in Kenyan school children, 80% in East Indian preschool-aged children, and 70% in East Indian adults ⁴. Certain risk factors increase the prevalence of vitamin B12 deficiency (see Table 1) ¹⁵. Dietary insufficiency, pernicious anemia (i.e., an autoimmune process that reduces available intrinsic factor and subsequent absorption of vitamin B12) ⁴ and long-term use of metformin or stomach acid-suppressing medications have been implicated in B12 deficiency ¹⁶, ¹⁷. A multicenter randomized controlled trial of 390 patients with diabetes mellitus showed that those taking 850 mg of metformin three times per day had an increased risk of vitamin B12 deficiency (number needed to harm = 14 per 4.3 years) and low vitamin B12 levels (number needed to harm = 9 per 4.3 years) vs. placebo ¹⁶. This effect increased with duration of metformin therapy, and patients had an unclear prophylactic supplementation response ¹⁶. A case-control study that compared 25,956 patients who had vitamin B12 deficiency with 184,199 control patients found a significantly increased risk of vitamin B12 deficiency in patients who had taken proton pump inhibitors or histamine H2 blockers for at least two years ¹⁷. In light of these findings, long-term use of these medications should be periodically reassessed, particularly in patients with other risk factors for vitamin B12 deficiency ¹⁶, ¹⁷.

Screening persons at average risk of vitamin B12 deficiency is not recommended ⁵. Screening for vitamin B12 deficiency should be considered in patients with risk factors, and diagnostic testing for vitamin B12 deficiency should be considered in those with suspected clinical signs and symptoms of vitamin B12 deficiency ¹⁸, ⁴, ¹⁹.

The recommended laboratory evaluation for patients with suspected vitamin B12 deficiency includes a complete blood count (CBC) and serum vitamin B12 level ¹⁸, ²⁰. A level of less than 150 pg per mL (111 pmol per L) is diagnostic for deficiency ¹⁸, ⁴. Serum vitamin B12 levels may be artificially elevated in patients with alcoholism, liver disease, or cancer because of decreased liver clearance of transport proteins and resultant higher circulating levels of vitamin B12; physicians should use caution when interpreting laboratory results in these patients ²¹, ²². In patients with a normal or low-normal serum vitamin B12 level, complete blood count results demonstrating macrocytosis, or suspected clinical manifestations, a serum methylmalonic acid (MMA) level is an appropriate next step ²³ and is a more direct measure of vitamin B12’s physiologic activity ¹⁸, ⁴. Although not clinically validated or available for widespread use, measurement of holotranscobalamin, the metabolically active form of vitamin B12, is an emerging method of detecting deficiency ⁵.

Typically, vitamin B12 deficiency is treated with intramuscular injections of cyanocobalamin or hydroxocobalamin, because this method bypasses any barriers to absorption. Hydroxocobalamin is usually the recommended option as it stays in the body for longer. Approximately 10% of the standard injectable dose of 1 mg is absorbed, which allows for rapid replacement in patients with severe deficiency or severe neurologic symptoms ⁷. Guidelines from the British Society for Haematology recommend injections three times per week for two weeks in patients without neurologic deficits ²³. If neurologic deficits are present, injections should be given every other day for up to three weeks or until no further improvement is noted.

However, high doses of oral vitamin B12 might also be effective ²⁴. A 2018 Cochrane review included three randomized controlled trials (RCTs) that compared very high doses (1,000–2,000 mcg) of oral with intramuscular vitamin B12 for vitamin B12 deficiency in a total of 153 participants ²⁵. The evidence from these studies, although of low quality, showed that the ability of high oral doses of vitamin B12 supplements to normalize serum vitamin B12 was similar to that of intramuscular vitamin B12. The British Society for Haematology recommends intramuscular vitamin B12 for severe deficiency and malabsorption syndromes, whereas oral replacement may be considered for patients with asymptomatic, mild disease with no absorption or compliance concerns ²³.

If vitamin B12 deficiency coexists with folate deficiency, vitamin B12 should be replaced first to prevent subacute combined degeneration of the spinal cord ⁴.

The British Society for Haematology does not recommend retesting vitamin B12 levels after treatment has been initiated, and no guidelines address the optimal interval for screening high-risk patients ²³. In general, patients with an irreversible cause should be treated indefinitely, whereas those with a reversible cause should be treated until the deficiency is corrected and symptoms resolve ⁴.

Figure 1. Vitamin B12 absorption and transport

Figure 2. Vitamin B12 deficiency pathophysiology

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Figure 3. Glossitis secondary to vitamin B12 deficiency anemia

Footnotes: (A) Generalized dryness of the tongue of a 61-year-old woman with vitamin B12 deficiency, with atrophy (blue arrowheads) and erythematous plaques (white arrowheads). (B) Normal appearance of the tongue 3 days after the patient received a single injection of vitamin B12.

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Table 1. Risk factors for vitamin B12 deficiency

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Table 2. Clinical and laboratory findings in vitamin B12 deficiency

General symptoms	Weight loss observed in most patients
	Low-grade fever occurs in one third of newly diagnosed patients and promptly disappears with treatment
Gastrointestinal symptoms	Smooth tongue (50% of patients) with loss of papillae. Changes in taste and loss of appetite
	Patients may report either constipation or having several semi-solid bowel movements daily
	Anorexia, nausea, vomiting, heartburn, pyrosis, flatulence and a sense of fullness
Brain	Altered mental status. Cognitive defects (“megaloblastic madness”): depression, mania, irritability, paranoia, delusions, lability
Sensory organs	Optic atrophy, anosmia, loss of taste, glossitis
Bone marrow	Hypercellular bone marrow
	Increased erythroid precursors
	Open, immature nuclear chromatin
	Dyssynchrony between maturation of cytoplasm and nuclei
	Giant bands, metamyelocytes
	Karyorrhexis, dysplasia
	Abnormal results on flow cytometry and cytogenetic analysis
Spinal cord	Myelopathy
	Spongy degeneration
	Paresthesias
	Loss of proprioception: vibration, position, ataxic gait, limb weakness/spasticity (hyperreflexia)
	Positive Romberg sign
	Lhermitte’s sign
	Segmental cutaneous sensory level
Autonomic nervous system	Postural hypotension
	Incontinence
	Impotence
Peripheral nervous system	Cutaneous sensory loss
	Hyporeflexia symmetric weakness
	Paresthesias
Genitourinary symptoms	Urinary retention and impaired micturition may occur because of spinal cord damage. This can predispose patients to urinary tract infections
Reproductive system	Infertility
Abnormalities in infants and children	Developmental delay or regression, permanent disability
	The patient does not smile
	Feeding difficulties
	Hypotonia, lethargy, coma
	Hyperirritability, convulsions, tremors, myoclonus
	Microcephaly
	Choreoathetoid movements, peripheral blood
	Macrocytic red cells, macro-ovalocytes
	Anisocytosis, fragmented forms
	Hypersegmented neutrophils
	Leukopenia, possible immature white cells
	Thrombocytopenia
	Pancytopenia
	Elevated lactate dehydrogenase level (extremes possible)
	Elevated indirect bilirubin and aspartate aminotransferase levels
	Decreased haptoglobin level
	Elevated levels of methylmalonic acid, homocysteine, or both

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What is vitamin B12?

Vitamin B12 also known as cobalamin or cyanocobalamin (man-made form of vitamin B12), is a water-soluble vitamin that is naturally present in some foods, added to others, and available as a dietary supplement and a prescription medication. Because vitamin B12 contains the mineral cobalt, compounds with vitamin B12 activity are collectively called “cobalamins” ²⁹. Methylcobalamin and 5-deoxyadenosylcobalamin are the metabolically “active” forms of vitamin B12 used by your body. However, two vitamin B-12 forms, hydroxycobalamin and cyanocobalamin, become biologically active after they are converted to methylcobalamin or 5-deoxyadenosylcobalamin ³⁰, ⁶, ³¹. Vitamin B-12 also helps prevent a type of anemia called megaloblastic anemia that makes people tired and weak.

Your body cannot make vitamin B12. Vitamin B-12 is synthesized only by bacteria ³². While present in animal products, including meats, fish, shellfish, dairy products, and eggs, it is absent in plant- based foods. People most at risk for vitamin B12 deficiency are vegans, as diets devoid of animal products will result in B12 deficiency. Based on the absorption of labeled vitamin B12 from some food products, such as chicken meat, rainbow trout, or eggs the bioavailability of vitamin B12 is generally assumed to be 40% or 50% for healthy adults without alteration of gastrointestinal functioning ³³. Bioavailability also varies by type of food source. For example, dairy products have a bioavailability of vitamin B12 three times higher than meat or fish ³⁴. Moreover, the bioavailability of vitamin B12 from dairy products is considerable ³⁵. Currently, to maintain a healthy hematological status and serum vitamin B12 levels, average daily intakes of vitamin B12 from food of 5.94 mcg for men and 3.78 mcg for women aged 20 and older have been recommended ². For children aged 2–19 years old, mean daily intakes of vitamin B12 from food range from 3.76 mcg to 4.55 mcg ³. The original estimates of dietary folate and vitamin B12 requirements and recommended dietary allowances (RDAs) were based on the amount needed to avoid manifest deficiency disorders (megaloblastic anemia, with sub-acute combined degeneration of the cord in the case of vitamin B12 deficiency) and on levels observed in populations. However, these levels do not essentially represent necessary requirements ².

Furthermore, vitamin B12 issues can be caused by taking some types of stomach acid blockers. Ruscin et al. ³⁶ illustrated the case of a 78-year-old non-vegetarian white woman with gastroesophageal reflux treated for long-term with histamine-2 (H2)-receptor antagonists and a proton-pump inhibitor (PPI). During treatment, her vitamin B12 dropped from normal values (413 pg/mL) to 256 pg/mL; methylmalonic acid (MMA) and homocysteine were elevated at 757 nmol/L and 27.3 micromol/L, respectively, serum folate was within the normal range (4.9 ng/mL), and serum creatinine was slightly elevated at 1.4 mg/dL ³⁶. In addition, no kidney dysfunction was present. After oral treatment with vitamin B12 (1000 mcg/day), her MMA (methylmalonic acid) and homocysteine concentrations decreased dramatically. The authors speculated vitamin B12 deficiency because of cobalamin malabsorption from food intake due to drug interference, suggesting vitamin B12 status monitoring in patients taking these medications for an extended time, particularly >4 years ³⁶.

Also, some people have an autoimmune or inflammatory condition of the stomach wall that degrade the proteins that aid vitamin B12 absorption can cause vitamin B12 deficiency. Vitamin B12 is bound to protein in food and must be released before it is absorbed ⁷. The process starts in the mouth when food is mixed with saliva. The freed vitamin B12 then binds with haptocorrin, a cobalamin-binding protein in the saliva ²⁴. More vitamin B12 is released from its food matrix by the activity of hydrochloric acid and gastric protease in the stomach, where it then binds to haptocorrin ²⁹. In the duodenum (the first part of your small intestine), digestive enzymes free the vitamin B12 from haptocorrin, and this freed vitamin B12 combines with intrinsic factor (IF), a transport and delivery binding protein secreted by the stomach’s parietal cells. The resulting complex is absorbed in the distal ileum (the last part of your small intestine) by receptor-mediated endocytosis ⁷. If vitamin B12 is added to fortified foods and dietary supplements, it is already in free form and therefore does not require the separation step ²⁴.

In the blood plasma, Vitamin B-12 is bound to transcobalamins 1 and 2. Transcobalamin 2 is responsible for delivering Vitamin B-12 to tissues. The liver stores large amounts of Vitamin B-12. Enterohepatic reabsorption helps retain Vitamin B-12. Liver Vitamin B-12 stores can normally sustain physiologic needs for 3 to 5 years if vitamin B12 intake stops (eg, in people who become vegans) and for months to 1 year if enterohepatic reabsorption capacity is absent.

Vitamin B12 status is typically assessed by measurements of serum or plasma vitamin B12 levels ²⁴. The cutoff between normal vitamin B12 levels and vitamin B12 deficiency varies by method and laboratory, but most laboratories define subnormal serum or plasma values as those lower than 200 or 250 pg/mL (148 or 185 pmol/L) ⁶. Levels of serum methylmalonic acid (MMA), a vitamin B12-associated metabolite, are the most sensitive markers of vitamin B12 status, and an MMA (methylmalonic acid) level greater than 0.271 micromol/L suggests vitamin B12 deficiency ³⁷, ³⁸, ⁵. However, methylmalonic acid (MMA) levels also rise with kidney insufficiency and tend to be higher in older adults ¹³, ³⁹, ³⁸. Another marker is total plasma homocysteine levels, which rise quickly as vitamin B12 status declines; a serum homocysteine level higher than 15 micromol/L, for example, suggests vitamin B12 deficiency ⁴⁰. However, this indicator has poor specificity because it is influenced by other factors, such as low folate levels and, especially, by declines in kidney function ³⁸. Experts suggest that if a patient’s serum vitamin B12 level is less than 150 pg/ml (111 pmol/L), the patient’s serum methylmalonic acid (MMA) levels should be checked to confirm a diagnosis of vitamin B12 deficiency ⁵, ¹³.

What does vitamin B12 do?

Vitamin B12 is required for the development, myelination, and function of the central nervous system; healthy red blood cell formation; and helps make DNA, the genetic material in all cells ⁷, ³⁰. Vitamin B12 functions as a cofactor for two enzymes, methionine synthase and L-methylmalonyl-CoA mutase (see more below) ⁷, ⁶, ³¹. Methionine synthase catalyzes the conversion of homocysteine to the essential amino acid methionine ²⁹, ⁶. Methionine is required for the formation of S-adenosylmethionine, a universal methyl donor for almost 100 different substrates, including DNA, RNA, proteins, and lipids ⁷, ³¹. L-methylmalonyl-CoA mutase converts L-methylmalonyl-CoA to succinyl-CoA in the metabolism of propionate, a short-chain fatty acid ⁶.

Vitamin B12 functions as a cofactor for methionine synthase

Methylcobalamin is required for the function of the folate-dependent enzyme, methionine synthase. The methionine synthase enzyme is required for the synthesis of the amino acid, methionine, from homocysteine. Methionine in turn is required for the synthesis of S-adenosylmethionine (SAMe), a methyl group donor used in many biological methylation reactions, including the methylation of a number of sites within DNA, RNA, and proteins ⁴¹. Aberrant methylation of DNA and proteins, which causes alterations in chromatin structure and gene expression, are a common feature of cancer cells. Inadequate function of methionine synthase can lead to an accumulation of homocysteine, which has been associated with increased risk of cardiovascular disease (Figure 4).

Figure 4. Vitamin B12 functions as a cofactor for methionine synthase

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Vitamin B12 functions as a cofactor for L-methylmalonyl-coenzyme A mutase

5-Deoxyadenosylcobalamin is required by the enzyme that catalyzes the conversion of L-methylmalonyl-coenzyme A to succinyl-coenzyme A (succinyl-CoA), which then enters the citric acid cycle (Figure 5). Succinyl-CoA plays an important role in the production of energy from lipids and proteins and is also required for the synthesis of hemoglobin, the oxygen-carrying pigment in red blood cells ⁴¹.

Figure 5. Vitamin B12 functions as a cofactor for L-methylmalonyl-coenzyme A mutase

[Source ⁴² ]

How much Vitamin B-12 do I need?

The amount of Vitamin B-12 you need each day depends on your age. Average daily recommended amounts for different ages are listed below in micrograms (mcg). Table 3 lists the current Recommended Dietary Allowance (RDA) for Vitamin B-12 in micrograms (mcg). For infants aged 0 to 12 months, the Food and Nutrition Board established an adequate intake (AI) for vitamin B-12 that is equivalent to the mean intake of Vitamin B-12 in healthy, breastfed infants.

• Recommended Dietary Allowance (RDA): average daily level of intake sufficient to meet the nutrient requirements of nearly all (97%–98%) healthy individuals.
• Adequate Intake (AI): established when evidence is insufficient to develop an RDA and is set at a level assumed to ensure nutritional adequacy.

Table 3. Vitamin B-12 Recommended Intake

Life Stage	Recommended Amount
Birth to 6 months	0.4 mcg
Infants 7–12 months	0.5 mcg
Children 1–3 years	0.9 mcg
Children 4–8 years	1.2 mcg
Children 9–13 years	1.8 mcg
Teens 14–18 years	2.4 mcg
Adults	2.4 mcg
Pregnant teens and women	2.6 mcg
Breastfeeding teens and women	2.8 mcg

[Source ⁴³ ].

What are food sources of vitamin B12?

Vitamin B12 is found naturally in a wide variety of foods of animal origin (such as fish, meat, poultry, eggs, and dairy products) and manufacturers add it to some fortified foods (e.g., fortified breakfast cereals and fortified nutritional yeasts) ⁷. Plant foods have no vitamin B12 unless they are fortified ⁴⁴. You can get recommended amounts of vitamin B12 by eating a variety of foods including the following:

• Fish, meat, poultry, eggs, milk, and other dairy products contain vitamin B12.
• Clams and beef liver are some of the best source of vitamin B12.
• Some breakfast cereals, nutritional yeasts, and other food products are fortified with vitamin B12.

The U.S. Department of Agriculture’s FoodData Central (https://fdc.nal.usda.gov) lists the nutrient content of many foods and provides a comprehensive list of foods containing vitamin B12 arranged by nutrient content (https://ods.od.nih.gov/pubs/usdandb/VitaminB12-Content.pdf) and by food name (https://ods.od.nih.gov/pubs/usdandb/VitaminB12-Food.pdf).

The average vitamin B12 level in the breast milk of women with vitamin B12 intakes above the RDA is 0.44 mcg/L ⁴⁵. The U.S. Food and Drug Administration (FDA) specifies that infant formulas sold in the United States must provide at least 0.15 mcg vitamin B12 per 100 kcal ⁴⁶.

The estimated bioavailability of vitamin B12 from food varies by vitamin B12 dose because absorption decreases drastically when the capacity of intrinsic factor is exceeded (at 1–2 mcg of vitamin B12) ⁴⁷. Bioavailability also varies by type of food source. For example, the bioavailability of vitamin B12 appears to be about three times higher in dairy products than in meat, fish, and poultry, and the bioavailability of vitamin B12 from dietary supplements is about 50% higher than that from food sources ⁴⁸.

A variety of foods and their vitamin B12 levels per serving are listed in Table 4.

Table 4. Selected Food Sources of Vitamin B-12

Food	Micrograms per serving	Percent DV*
Beef liver, cooked, pan-fried, 3 ounces	70.7	2944
Clams (without shells), cooked, 3 ounces	17	708
Tuna, bluefin, cooked, dry heat, 3 ounces	9.3	385
Nutritional yeast, fortified, from several brands (check label), about ¼ cup	8.3 to 24	346 to 1,000
Salmon, Atlantic, cooked, 3 ounces	2.6	108
Beef, ground, 85% lean meat/15% fat, pan-browned, 3 ounces	2.4	100
Milk, 2% milkfat, 1 cup	1.3	54
Yogurt, plain, fat free, 6-ounce container	1	43
Breakfast cereals, fortified with 25% of the DV for vitamin B12, 1 serving	0.6	25
Cheese, cheddar, 1½ ounces	0.5	19
Egg, whole, cooked, 1 large	0.5	19
Turkey, breast meat, roasted, 3 ounces	0.3	14
Tempeh, 1/2 cup	0.1	3
Banana, 1 medium	0	0
Bread, whole-wheat, 1 slice	0	0
Strawberries, raw, halved, 1/2 cup	0	0
Beans, kidney, boiled, 1/2 cup	0	0
Spinach, boiled, drained, 1/2 cup	0	0

Footnote: *DV = Daily Value. DVs were developed by the U.S. Food and Drug Administration (FDA) to help consumers determine the level of various nutrients in a standard serving of food in relation to their approximate requirement for it. The DV for Vitamin B-12 is 6.0 mcg. However, the FDA does not require food labels to list Vitamin B-12 content unless a food has been fortified with this nutrient. Foods providing 20% or more of the DV are considered to be high sources of a nutrient, but foods providing lower percentages of the DV also contribute to a healthful diet.

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Who are at risk of vitamin B12 deficiency?

The following people are among those most likely to be vitamin B12 deficient.

Older adults

Depending on the definition used, between 3% and 43% of community-dwelling older adults, especially those with atrophic gastritis (chronic inflammation and thinning of your stomach), have vitamin B12 deficiency based on serum vitamin B12 levels ⁵⁰, ⁵¹. The vitamin B12 deficiency rate at a cutoff of less than 211 mcg/L (156 pmol/L) at admission to a long-term care facility, according to one study, was 14%, and 38% of these older adults had levels lower than 407 pg/mL (300 pmol/L) ⁵¹.

Conditions associated with vitamin B12 deficiency include pernicious anemia, present in about 15% to 25% of older adults with vitamin B12 deficiency ²⁸. Atrophic gastritis, an autoimmune condition affecting 2% of the general population but 8–9% of adults aged 65 and older, decreases production of intrinsic factor and secretion of hydrochloric acid in the stomach and thus decreases absorption of vitamin B12 ²⁸, ⁵². A third condition associated with vitamin B12 deficiency in older adults is Helicobacter pylori infection, possibly because this bacterium causes inflammation that leads to malabsorption of vitamin B12 from food ⁵³.

Individuals with pernicious anemia

Pernicious anemia is an irreversible autoimmune disease that affects the gastric mucosa and results in gastric atrophy ⁵⁴. This disease leads to attacks on parietal cells in the stomach, resulting in failure to produce intrinsic factor (IF) and malabsorption of dietary vitamin B12, recycled biliary vitamin B12, and free vitamin B12 ⁴⁰, ³⁸. Therefore, without treatment, pernicious anemia causes vitamin B12 deficiency, even in the presence of adequate vitamin B12 intakes.

Pernicious anemia is the most common cause of clinically evident vitamin B12 deficiency around the world ⁴⁰, ⁵⁴. The incidence of pernicious anemia in the United States is an estimated 151 per 100,000, and this condition is more common in women and in people of European ancestry ⁵⁴.

Individuals with gastrointestinal disorders

Individuals with stomach and small intestine disorders, such as celiac disease and Crohn’s disease, may be unable to absorb enough vitamin B12 from food to maintain healthy body stores ⁵⁵. But although rates of vitamin B12 deficiency are higher in people with celiac disease than other people ⁵⁶, the evidence for whether rates of vitamin B12 deficiency are higher in people with Crohn’s disease is mixed ⁵⁷, ⁵⁸. Vitamin B12 deficiency in people with Crohn’s disease is typically treated with intramuscular cobalamin injections, but high doses of oral cyanocobalamin therapy (e.g., 1,000 mcg/day) might be equally effective ⁵⁹.

Individuals who have had gastrointestinal surgery

Surgical procedures in the gastrointestinal tract, such as for weight loss (bariatric surgery) or to remove all or part of the stomach (gastrectomy), can cause a complete or partial loss of cells that secrete hydrochloric acid and cells that secrete intrinsic factor (IF) ⁶⁰, ⁶¹. Thus, these procedures reduce the amount of vitamin B12, particularly food-bound vitamin B12, that the body absorbs ⁶⁰, ⁶¹. High doses (1,000 mcg/day) of oral methylcobalamin supplements appear to be as effective as hydroxycobalamin injections in normalizing vitamin B12 values in patients who have undergone Roux-en-Y gastric bypass surgery ⁶².

Vegetarians

Vegans who consume no animal products and vegetarians who consume some animal products (e.g., dairy products, eggs, or both) but not meat have a higher risk of developing vitamin B12 deficiency because natural food sources of vitamin B12 are limited to animal foods ⁶³. Consumption of foods fortified with vitamin B12 (such as fortified nutritional yeasts) as well as vitamin B12 supplements can substantially reduce the risk of deficiency ⁶³.

Infants of vegan women

Exclusively breastfed infants of women who consume no animal products might have very limited reserves of vitamin B12 and can develop vitamin B12 deficiency, sometimes very early in life ⁶⁴. The infant’s vitamin B12 deficiency can be severe, especially if the mother’s vitamin B12 deficiency is severe or caused by pernicious anemia; sometimes, the mother’s own vitamin B12 deficiency is clinically mild and not recognized. Undetected and untreated vitamin B12 deficiency in infants can result in neurological damage, failure to thrive, developmental delays, and anemia ⁶⁴, ⁶⁵. The reasons include the small amounts of vitamin B12 in the breast milk of vegan mothers as well as the limited amounts of vitamin B12 crossing the placenta in these women during fetal development.

What is pernicious anemia?

Pernicious anemia is a condition in which the body can’t make enough healthy red blood cells because of severe vitamin B12 deficiency due to an autoimmune inflammation of the stomach (autoimmune gastritis), resulting in destruction of stomach parietal cells by one’s own antibodies resulting in intrinsic factor (IF) deficiency ⁶⁶. Progressive destruction of the parietal cells that line the stomach cause decreased secretion of acid and enzymes required to release food bound vitamin B12. Antibodies to intrinsic factor (IFA) bind to IF preventing formation of the IF-B12 complex, further inhibiting vitamin B12 absorption. Without enough vitamin B12, your red blood cells don’t divide normally and are too large (megaloblasts). These changes occur because vitamin B12 is necessary for DNA synthesis ⁶⁷. In addition to megaloblasts, hypersegmented neutrophils are also present. The large red blood cells may have trouble getting out of the bone marrow—a sponge-like tissue inside the bones where blood cells are made. Megaloblastic anemia is characterized by large nucleated red blood cell precursors called megaloblasts in the bone marrow ⁶⁷. Without enough red blood cells to carry oxygen to your body, you may feel tired and weak. Severe or long-lasting pernicious anemia can damage the heart, brain, and other organs in the body. Note that the causes of megaloblastic anemia other than vitamin B12 deficiency caused by impaired intrinsic factor (IF) production can include folic acid deficiency, altered pH in the small intestine, and lack of absorption of vitamin B12 complexes in the terminal ileum. Thus, pernicious anemia must be differentiated from other disorders that interfere with the absorption and metabolism of vitamin B12.

Pernicious anemia also can cause other problems, such as nerve damage, neurological problems (such as memory loss), and digestive tract problems. People who have pernicious anemia also may be at higher risk for weakened bone strength and stomach cancer. Pernicious anemia is frequently presenting with other autoimmune conditions such as autoimmune thyroid disease, type 1 diabetes, and vitiligo ⁶⁸.

Vitamin B12 is a nutrient found in some foods. The body needs this nutrient to make healthy red blood cells and to keep its nervous system working properly.

People who have pernicious anemia can’t absorb enough vitamin B12 from food. This is because they lack intrinsic factor (IF), a protein made in the stomach. A lack of this protein leads to vitamin B12 deficiency.

Pernicious anemia accounts for 20%‐50% of the vitamin B12 deficiency in adults ⁶⁹ and is associated with autoimmune gastritis, resulting in the destruction of gastric parietal cells and the associated lack of intrinsic factor ⁷⁰. The prevalence of pernicious anemia is estimated at 10‐50 per 100,000 persons, among North Europeans and Caucasian Americans. The prevalence of pernicious anemia in Japan is rare, 1‐5 per 100 000 persons ⁷¹, compared with the West. Pernicious anemia is caused by autoimmune metaplastic atrophic gastritis, which predominantly manifests in the stomach body and fundus. In pernicious anemia, antigastric parietal cell autoantibodies are detected specifically against the hydrogen potassium adenosine triphosphatase (H+/K+‐ATPase) proton pump ⁷². Helicobacter pylori (H. pylori) are not generally considered to be associated with autoimmune metaplastic atrophic gastritis. However, Hershko et al. ⁷³ have reported that H. pylori might serve as a trigger of autoimmune metaplastic atrophic gastritis and pernicious anemia, based on their observation that the prevalence of H. pylori infection was 87.5% in patients under 20 years of age. In addition, one theory regarding the initiating event of autoimmune metaplastic atrophic gastritis is molecular mimicry between H. pylori antigens and gastric H+/K+−ATPase ⁷⁴.

The destruction of parietal cells leads to decreased acid production and intrinsic factor secretion, and autoantibodies against intrinsic factor inhibit the absorption of vitamin B12. As a result, gastrin secretion from antral G cells increases, and hypergastrinemia induces proliferation of oxyntic mucosal cells including enterochromaffin‐like cells and parietal cells ⁷⁵. The clinical manifestations are similar to other vitamin B12 deficiencies, but pernicious anemia is sometimes associated with other autoimmune diseases such as type 1 diabetes, autoimmune thyroiditis, and Addison’s disease. Sensitivity and specificity of the anti‐intrinsic factor antibody test were 50%‐70%, and greater than 95%, respectively ⁷⁶. Sensitivity and specificity of the antigastric parietal cell antibody test were more than 90% and 50%, respectively ⁷⁷. The treatment for pernicious anemia is lifelong administration of vitamin B12. Patients with pernicious anemia are at high risk of developing gastric adenocarcinoma and carcinoid tumors ⁷⁸. Significant risk factors for the development of gastric carcinoma in autoimmune metaplastic atrophic gastritis include the presence of pernicious anemia, severity of mucosal atrophy, intestinal metaplasia, disease duration, and over 50 years of age ⁷⁵. Periodic stomach examinations are recommended for patients with pernicious anemia.

Other conditions and factors also can cause vitamin B12 deficiency. Examples include infections, surgery, medicines, and diet. Technically, the term “pernicious anemia” refers to vitamin B12 deficiency due to a lack of intrinsic factor. Often though, vitamin B12 deficiency due to other causes also is called pernicious anemia.

Pernicious anemia is one of two major types of “macrocystic” or “megaloblastic” anemia. These terms refer to anemia in which the red blood cells are larger than normal. The other major type of macrocystic anemia is caused by folic acid deficiency.

Rarely, children are born with an inherited disorder that prevents their bodies from making intrinsic factor. This disorder is called congenital pernicious anemia ⁷⁹. This condition is quite rare and distinguishable from the usual form of pernicious anemia due to the early age of onset and the absence of gastric corpus atrophy.

Causes of pernicious anemia

Pernicious anemia is an autoimmune disorder. Typically, pernicious anemia is associated with the presence of autoantibodies against intrinsic factor (anti-intrinsic factor antibody or IFA) and anti-parietal cell antibodies (PCA), thus supporting the autoimmune origin of this condition ⁸⁰. Anti-parietal cell antibodies (PCA) are seen in up to 85% of patients with pernicious anemia ⁸¹. However, the anti-parietal cell antibody (PCA) is not specific for pernicious anemia and can be seen in 3 to 10% of normal healthy populations without any evidence of megaloblastic anemia ⁸¹. Antibodies against parietal cells (PCA) are class M, G, and A immunoglobulins directed towards the alpha and beta subunits of the gastric proton pump (hydrogen-potassium ATP-ase) ⁸². Anti-intrinsic factor antibodies (IFA) are seen in 40 to 60% of the patients with pernicious anemia and are highly specific for the disease ⁸¹. Antibodies against intrinsic factor (IFA) are class G immunoglobulins that target the binding site for cobalamin (type I) or the binding site for ileal epithelial mucosa (type II) ⁸³. These autoantibodies are released from plasma cells activated by autoreactive CD4+ T cell lymphocytes in perigastric lymph nodes ⁸⁴. These triggered CD4+ T cells target the proton pump ATPases, which leads to their immune destruction ⁸¹. Atrophic gastritis with loss of parietal cells and subsequent intrinsic factor (IF) deficiency develops. This leads to vitamin B12 deficiency and the onset of symptoms associated with pernicious anemia. Gastric dendritic cells are responsible for the activation of lymphocytes that lead to the production of these antibodies. The cause and mechanism of dendritic cell activation are not yet clarified ⁸¹. Some research studies suggest Helicobacter pylori (H. pylori) infection is a trigger in genetically susceptible individuals ⁸⁵. The studies propose molecular mimicry and immune cross-reactivity between the proton pump ATPase and the H. pylori antigens as a triggering event ⁸⁵. A 2017 review evaluating H. pylori antigenic mimicry stated that these antigens play an important role in the induction of humoral and cellular immune responses, which may predispose patients to pathological inflammatory responses ⁸⁶.

These circulating anti-intrinsic factor antibodies (IFA) and anti-parietal cell antibodies (PCA) cause autoimmune chronic atrophic gastritis with parietal cell loss and eventual vitamin B12 deficiency ⁸¹. However, autoimmune gastritis-associated gastric corpus atrophy may progress without developing pernicious anemia ⁷⁹.

Pernicious anemia can be associated with other autoimmune diseases and in patients with polyglandular autoimmune disorders. Autoimmune diseases associated with pernicious anemia include type 1 diabetes (3 to 4%), vitiligo (2 to 8%), and autoimmune thyroid disease (3 to 32%) ⁸⁰. Type III polyglandular autoimmune syndrome is characterized by the presence of autoimmune thyroiditis, vitiligo, alopecia, type 1A diabetes mellitus, pernicious anemia, and chronic atrophic gastritis ⁸⁷. HLA alleles are thought to play a role in the pathogenesis of these autoimmune disorders, but the mechanism is not entirely understood. HLA-DRB1/03 and HLA-DRB1/04 alleles may predispose to autoimmune gastritis and subsequent pernicious anemia ⁸⁰.

There is an overlap in patients infected with Helicobacter pylori (H. pylori) and the development of chronic atrophic gastritis associated with pernicious anemia ⁸⁸. Researchers propose H. pylori peptide-induced gastric T-cell proliferation as the cause of pernicious anemia in some patients ⁸⁵. They were able to demonstrate the presence of activated T cells in the gastric mucosa of patients with autoimmune chronic atrophic gastritis and H. pylori infection. These T cells reacted to both hydrogen-potassium-ATPase and H. pylori ⁸⁵. Recent experimental and clinical data suggest long-standing H. pylori infection plays a pivotal role in developing atrophic gastritis and subsequent pernicious anemia; however, convincing data to support H. pylori infection as a definite cause of pernicious anemia is still lacking ⁸⁰. Molecular analyses have revealed hydrogen-potassium-ATPase epitopes that are similar to, or cross-reactive with, epitopes of H. pylori antigens. Thus suggesting that in genetically susceptible individuals, H. pylori infection can trigger gastric autoimmunity via molecular mimicry ⁸⁵.

A heritable form of pernicious anemia called “childhood pernicious anemia” is seen in children with a genetic defect that leads to decreased IF production or abnormal IF formation ⁷⁹. This condition is quite rare and distinguishable from the usual form of pernicious anemia due to the early age of onset and the absence of gastric corpus atrophy.

Risk Factors for pernicious anemia

Pernicious anemia is more common in people of Northern European and African descent than in other ethnic groups.

Older people also are at higher risk for the condition. This is mainly due to a lack of stomach acid and intrinsic factor, which prevents the small intestine from absorbing vitamin B12. As people grow older, they tend to make less stomach acid.

Pernicious anemia also can occur in younger people and other populations. You’re at higher risk for pernicious anemia if you:

• Have a family history of the condition.
• Have had part or all of your stomach surgically removed. The stomach makes intrinsic factor. This protein helps your body absorb vitamin B12.
• Have an autoimmune disorder that involves the endocrine glands, such as Addison’s disease, type 1 diabetes, Graves’ disease, or vitiligo. Research suggests a link may exist between these autoimmune disorders and pernicious anemia that’s caused by an autoimmune response.
• Have had part or all of your small intestine surgically removed. The small intestine is where vitamin B12 is absorbed.
• Have certain intestinal diseases or other disorders that may prevent your body from properly absorbing vitamin B12. Examples include Crohn’s disease, intestinal infections, and HIV.
• Take medicines that prevent your body from properly absorbing vitamin B12. Examples of such medicines include antibiotics and certain seizure medicines.
• Are a strict vegetarian who doesn’t eat any animal or dairy products and doesn’t take a vitamin B12 supplement, or if you eat poorly overall.

Pernicious anemia signs and symptoms

The onset of pernicious anemia usually is insidious and vague. The main signs and symptoms of pernicious anemia are hematological and neurological consequences of vitamin B12 deficiency, and both require several years for their development. The classic presentation consists of a triad of jaundice, glossitis, and myeloneuropathy ⁸⁹. However, with advances in clinical detection and often routine laboratory testing, this classic triad of jaundice, glossitis, and myeloneuropathy is now a rarity. Many pernicious anemia patients are incidentally noted to have macrocytic anemia and are ultimately diagnosed with this condition. Others may either present with symptoms attributable to anemia, such as lethargy and inability to concentrate, or with symptoms attributable to neuronal damage, such as paresthesias, imbalance, and spasticity ⁸¹.

General symptoms

Weight loss of 10-15 lb occurs in about 50% of patients and probably is due to anorexia, which is observed in most patients. Low-grade fever occurs in one third of newly diagnosed patients and promptly disappears with treatment.

Heart symptoms

Individuals with pernicious anemia often tolerate the anemia well, and many are ambulatory with hematocrit levels in the mid-teens. However, the cardiac output is usually increased when hematocrit levels fall below 20%, with associated accerations in heart rate. Congestive heart failure and coronary insufficiency can occur, most particularly in patients with preexisting heart disease.

Gastrointestinal symptoms

Approximately 50% of patients with pernicious anemia develop atrophic glossitis, presenting with a smooth tongue that may be painful and beefy red, with loss of papillae that is usually most marked along the edges of the tongue ⁹⁰. These patients report burning or soreness, most particularly on the anterior third of the tongue, associated with changes in taste and loss of appetite ²⁷.

Patients may report either constipation or having several semisolid bowel movements daily. These symptoms have been attributed to megaloblastic changes of the cells of the intestinal mucosa.

Nonspecific gastrointestinal symptoms are not unusual and include anorexia, nausea, vomiting, heartburn, pyrosis, flatulence, and a sense of fullness ⁹¹. Rarely, patients present with severe abdominal pain associated with abdominal rigidity; this has been attributed to spinal cord pathology. Venkatesh and colleagues ⁹² report the case of a patient who presented with epigastric pain, diarrhea, and vomiting and was found to have thrombosis of the portal, superior mesenteric, and splenic veins due to hyperhomocysteinemia secondary to pernicious anemia.

Neurologic symptoms

The most common neurologic symptoms in vitamin B12 deficiency include paresthesias, weakness, clumsiness, and an unsteady gait. The last two symptoms are exacerbated in dark environments due to the loss of visual cues that patients often rely on, in concert with the loss of proprioception. These neurologic symptoms are due to myelin degeneration and loss of nerve fibers in the dorsal and lateral columns of the spinal cord and cerebral cortex (subacute combined degeneration).

Neurologic symptoms and findings may be present in the absence of anemia. This is more common in patients taking folic acid or on a high-folate diet.

Older patients may present with symptoms suggesting senile dementia or Alzheimer disease; memory loss, irritability, and personality changes are commonplace ⁸¹. Common psychiatric manifestations include depression, mania, chronic fatigue syndrome, and psychosis ⁷⁹. Cognitive symptoms include memory impairment, attention deficit, and dementia ⁷⁹. So-called megaloblastic madness—delusions, hallucinations, outbursts, and paranoid schizophrenic ideation—is less common. Identifying the cause is important because significant reversal of these symptoms and findings can occur with vitamin B12 administration.

While neurologic symptoms usually occur in the elderly, they can rarely occur in the young ⁹³. Kocaoglu et al. ⁹⁴ reported a case of vitamin B12 deficiency and cerebral atrophy in a 12-month-old infant whose development had slowed since 6 months of age; the infant was exclusively breastfed and his mother was a long-time vegetarian. Neurologic recovery began within days after the infant received an intramuscular cobalamin injection.

Genitourinary symptoms

Urinary retention and impaired micturition may occur because of spinal cord damage. This can predispose patients to urinary tract infections.

Symptoms of thrombotic complications

A study of four patients revealed that pernicious anemia can lead to hyperhomocysteinemia that is significant enough to lead to venous thrombosis, even in the absence of any other risk factors for thromboembolism ⁹⁵.

Pernicious anemia complications

One of the most dreaded complications of pernicious anemia is the development of gastric cancer ⁹⁶. According to the Surveillance, Epidemiology, and End Results (SEER) Medicare database, patients with pernicious anemia are at higher risk for:

• Gastric adenocarcinoma
• Gastric carcinoid tumors
• Tonsillar cancer
• Hypopharyngeal cancer
• Esophageal squamous cell carcinoma
• Myeloma
• Acute myeloid leukemia
• Myelodysplastic syndrome

A 2013 systematic review revealed an incidence rate of 0.27% per patient-year for gastric cancer in patients with pernicious anemia, with a nearly sevenfold increased risk of gastric cancer in these patients ⁹⁷. For this reason, an upper gastrointestinal endoscopy is recommended when pernicious anemia is diagnosed ⁸⁰. Repeat endoscopies without any evidence of gastrointestinal symptoms are generally not recommended. Surveillance endoscopy every three years is recommended by some experts in patients with documented evidence of advanced chronic autoimmune atrophic gastritis ⁹⁸.

Pernicious anemia prevention

Because an increased familial incidence of pernicious anemia exists, family members should be aware that they are at greater risk of developing this disease and should seek medical attention promptly if they develop anemia or mental and neurologic symptoms ⁹⁹. Monitor siblings and children of patients with a hereditary abnormality of vitamin B12 deficiency for evidence of the specific defect in cobalamin transport or metabolism.

Determine whether vitamin B12 deficiency is the cause in patients who recently developed evidence of mental deterioration.

Prophylactically treat patients with vitamin B12 when they have undergone total gastrectomy, bypass procedures for weight reduction, ileectomy, pancreatectomy, or when they have atrophic gastritis or chronic inflammatory disease of the ileum ⁹⁹.

Strict vegetarians should continue supplementary vitamin B12, particularly during pregnancy and while nursing a newborn infant ⁹⁹.

Elderly people are at risk for developing pernicious anemia due to achlorhydria. Therefore, serum vitamin B-12 levels should be checked. If low or if cobalamin deficiency is suspected, they should be treated with vitamin B-12 supplementation.

Pernicious anemia diagnosis

The workup for pernicious anemia may include the following ¹⁰⁰:

• Complete blood cell count (CBC)
• Peripheral blood smear
• Indirect bilirubin and lactate dehydrogenase assays
• Evaluation of gastric secretions. Total gastric secretions are decreased to about 10% of the reference range. Most patients with pernicious anemia are achlorhydric, even with histamine stimulation. Intrinsic factor (IF) is either absent or markedly decreased.
• Serum vitamin B12 (cobalamin), folic acid, methylmalonic acid (MMA) and homocysteine assays.
  • Serum cobalamin reference ranges may vary slightly among different laboratories, but are generally from 200–900 pg/mL. Values of 180-250 pg/mL are considered bordeline, while less than 150 pg/mL is considered diagnostic of vitamin B12 deficiency. In these cases, elevated levels of methylmalonic acid (MMA) and total homocysteine can confirm the diagnosis ¹⁸.
  • The serum cobalamin level is usually low in patients with pernicious anemia. However, up to a third of patients can present with normal vitamin B12 levels and normocytic anemia, which often delays diagnosis ¹⁰¹. Certain patients with other forms of cobalamin deficiency, such as some inborn forms of cobalamin deficiency, transcobalamin 2 deficiency, and cobalamin deficiency due to nitrous oxide, can also present with normal serum cobalamin levels.
  • Serum cobalamin levels may also be low in patients with no clinical or identifiable metabolic abnormality ²³. Causes of falsely low serum cobalamin levels inclue the following:
    • Pregnancy
    • Oral contraceptives and hormone replacement therapy
    • Multiple myeloma
    • Transcobalamin 1 (TC1) deficiency
    • Severe folic acid deficiency
    • Ascorbic acid in high doses
  • A serum folic acid assay is useful for ruling out folic acid deficiency. The reference range is 2.5-20 ng/mL. Blood should be drawn before patients have a single hospital meal since food can restore serum folic acid levels to normal. Red blood cell folic acid level is not influenced by food.
• Levels of antibodies against intrinsic factor (IF) or the cells which make intrinsic factor.
• Schilling test (no longer available in most medical centers)
• A clinical trial of vitamin B12
• Bone marrow aspiration and biopsy (only needed if diagnosis is unclear)

Complete blood cell count (CBC) and peripheral blood smear may show the mean corpuscular volume (MCV) and mean cell hemoglobin (MCH) are increased, with a mean corpuscular hemoglobin concentration (MCHC) within the reference range ¹⁰⁰. However, up to 30% of patients with pernicious anemia may lack macrocytosis ⁷⁹. A normal MCV (mean corpuscular volume) does not rule out megaloblastic anemia, and pathognomonic megaloblasts are rarely seen. The hematocrit must fall by 20% before megaloblasts appear in the blood ⁸⁹. Anisocytosis and an increase in the red cell distribution width is the earliest measurable change in red cell indices to hint toward the diagnosis ⁸⁹.

The peripheral blood usually shows a macrocytic anemia with a mild leukopenia and thrombocytopenia. The leukopenia and thrombocytopenia usually parallel the severity of the anemia. The peripheral smear shows oval macrocytes, hypersegmented granulocytes, and anisopoikilocytosis. In severe anemia, red blood cell inclusions may include Howell-Jolly bodies, Cabot rings, and punctate basophilia. The macrocytosis can be obscured by the coexistence of iron deficiency, thalassemia minor, or inflammatory

The indirect bilirubin level may be elevated because pernicious anemia is a hemolytic disorder associated with increased turnover of bilirubin ¹⁰⁰. The serum lactate dehydrogenase (LDH) concentration usually is markedly increased ¹⁰⁰. Increased values for other red blood cells, enzymes, and serum iron saturation also are observed. The serum potassium, cholesterol, and skeletal alkaline phosphatase often are decreased.

A significantly decreased serum cobalamin level along with a typical clinical presentation, a characteristic peripheral smear, and an increased indirect bilirubin and LDH level is sufficient evidence for the diagnosis of a megaloblastic anemia.

Serum methylmalonic acid and homocysteine tests are important confirmatory tests but are not first-line tests. Elevated serum methylmalonic acid and homocysteine levels are found in patients with pernicious anemia. They probably are the most reliable test for cobalamin deficiency in patients who do not have a congenital metabolism disorder. In the absence of an inborn error of methylmalonic acid metabolism, methylmalonic aciduria is a sign of cobalamin deficiency.

Table 5. Serum methylmalonic acid (MMA) and homocysteine values used in differentiating between vitamin B12 deficiency and folic acid deficiency

Patient Condition	Methylmalonic Acid	Homocysteine
Healthy	Normal	Normal
Vitamin B12 deficiency	Elevated	Elevated
Folate deficiency	Normal	Elevated

[Source ¹⁰² ]

Testing for B12 Deficiency

A B12 level below 200 pg/mL (ng/L) is consistent with vitamin B12 deficiency ⁸⁸. Levels between 200 to 400 pg/mL are considered borderline ⁸¹. Serum B12 measurement alone has poor sensitivity and specificity for detecting B12 deficiency ⁸⁹. In patients with pernicious anemia, this level will be falsely elevated in 22 to 35% of the patients due to the interaction of IF antibody (IFA) with the “IF reagent” used to detect B12 levels in current assays ⁸¹. Falsely low serum vitamin B12 levels can occur in patients with underlying multiple myeloma and pregnancy ²³, ⁸¹.

Methylmalonic acid (MMA) and homocysteine levels can be obtained in patients when vitamin B12 levels are borderline or nondiagnostic to confirm the diagnosis of B12 deficiency ¹⁰³. These assays are considered more sensitive and specific for detecting B12 deficiency when compared to serum B12 levels ⁸⁹. Methylmalonic acid (MMA) can also help differentiate between vitamin B12 and folate deficiency, as it is elevated in vitamin B12 deficiency but not in folate deficiency ⁸⁹. Homocysteine levels are elevated in folate deficiency, vitamin B6 deficiency, and patients with hypothyroidism ²³. MMA level can be falsely elevated in patients with bacterial overgrowth, especially when there are blind loops of the bowel (following gastric surgery) ⁸⁹. Both levels can be falsely elevated in patients with renal failure ⁸⁹, ²³.

Serum holotranscobalamin (holoTC) level measures the metabolically active fraction of serum vitamin B12 and is considered a more accurate test for detecting B12 deficiency ²³. Transcobalamin is a transport protein that binds only 10 to 30% of the total plasma B12; however, this constitutes all of the “active fraction” used for metabolic activity ⁸⁹. Limitations of this test include a large window with indeterminate values ¹⁰³. In addition, according to one study, approximately 63% of patients with low holoTC levels had normal methylmalonic acid levels, raising concerns regarding the utility of this test as a true measure of B12 deficiency ¹⁰³. A 2013 study measuring the utility of biomarkers for B12 deficiency compared serum B12 levels to holotranscobalamin and recommended holotranscobalamin as the initial screening test for the detection of B12 deficiency, followed by MMA levels ¹⁰⁴. They also suggested that an indeterminate holotranscobalamin level between 23 and 75 pmol should be followed by methylmalonic acid testing. Of note, this study was conducted in patients with normal renal function.

Definitive Testing for Pernicious Anemia

Traditionally, vitamin B12 absorption was measured using the Schilling test. This test is now considered obsolete, and there is no available assay for detecting B12 absorption at this time ⁸⁹. In the absence of reliable B12 absorption assays, definitive testing for pernicious anemia relies on the detection of circulating antibodies to intrinsic factors (IFA) and gastric parietal cells (PCA).

Demonstration of circulating intrinsic factor autoantibodies is almost diagnostic of type A (autoimmune) gastritis and pernicious anemia. Intrinsic factor (IF) antibodies are specific for this disorder and can be used to confirm the diagnosis ⁸⁹. There are two types of IF antibodies (IFA). Type 1 IF antibodies block binding of vitamin B12 to intrinsic factor and are found in 70% to 90% of patients with pernicious anemia. Type 2 IF antibodies prevent attachment of the vitamin B12–IF complex to ileal receptors and are present in approximately 35% to 50% of patients with pernicious anemia; they rarely occur in the absence of type 1 IF antibodies. Both type 1 and type 2 antibodies are detected more often in gastric juice than in the serum ¹⁰⁵.

In one case report, the presence of antibodies to intrinsic factors (IFA) was used to diagnose vitamin B12 deficiency in a patient with severe leukoencephalopathy ¹⁰⁶. Interestingly, serum vitamin B12, homocysteine, and methylmalonic acid levels were normal. The patient responded to intensive cobalamin therapy ¹⁰⁶.

Parietal cell antibodies occurs in 90% of patients with pernicious anemia. However, antibodies to parietal cells (PCA) are not specific for pernicious anemia ⁸¹. Some experts advise against routine testing for antibodies to parietal cells (PCA); others recommend routine testing with anti-IF antibodies (IFA) because the combined sensitivity for pernicious anemia approaches 73% ⁶⁷, ²³. Dual testing for  intrinsic factors antibodies (IFA) and parietal cell antibodies (PCA) with proof of atrophic gastritis is 100% specific for pernicious anemia ⁸¹.

In ambiguous cases, a bone marrow biopsy showing megaloblastic erythropoiesis and arrested maturation of myeloid precursor cells will establish the diagnosis ⁶⁷. An alternative approach in difficult cases is to establish the presence of atrophic gastritis with endoscopic evaluation and biopsy and/or showing the presence of hypergastrinemia  ⁶⁷. In rare situations, an empiric trial of vitamin B12 replacement can be used to make the diagnosis  ⁶⁷. In this scenario, a rise in the reticulocyte count (which occurs within 5 to 14 days) confirms the diagnosis.

Alternative and new approaches to the diagnosis of pernicious anemia are under evaluation. One of these is a newer cobalamin absorption test, which has its basis in measuring the change in serum holotranscobalamin following oral ingestion of non-radiolabeled cobalamin. Another approach has been described using accelerator mass spectrometry to quantify 14C in the blood following an orally administered dose of [14C]-cyanocobalamin ⁸⁹. Recently an ELISA test measuring serum concentration of human IF has been developed and may prove to be an alternate measure of impaired IF production/absorption ¹⁰⁷.

Once the diagnosis of pernicious anemia is established, confirmatory testing with gastroscopy and histologic assessment of the gastric mucosa to assess for the presence of atrophic gastritis is indicated ⁷⁹. Pernicious anemia is recognized as a late-stage complication of autoimmune gastritis, with an increased risk of gastric cancers in this population ⁷⁹. Therefore, a new diagnosis of pernicious anemia warrants endoscopy with biopsies to detect the presence of atrophic gastritis and to rule out gastric cancers ⁹⁸. The presence of intestinal metaplasia on gastric biopsy confers a diagnosis of atrophic gastritis ⁹⁸. Pale gastric mucosa and increased visibility of vasculature are typical endoscopic features of atrophic gastritis. With metaplasia, light-blue crests and white opaque fields are present ⁹⁸.

Recent advances in endoscopic techniques have led to the development of an endoscopic grading of gastric intestinal metaplasia (EGGIM) using noninvasive techniques to assess the presence of metaplasia during endoscopy without any need for biopsies. This system has shown acceptable sensitivity and specificity compared to biopsies and can be used when evaluating patients with pernicious anemia ⁷⁹. In a cross-sectional study of 210 patients with atrophic gastritis, endoscopic grading of gastric intestinal metaplasia (EGGIM) was found to reliably identify more than 90% of patients with gastric corpus intestinal metaplasia. This method was shown to overestimate intestinal metaplasia when pseudopyloric metaplasia was present ⁷⁹.

Additional testing

Testing for iron deficiency is indicated for all patients with pernicious anemia ⁷⁹. Up to 20% of the patients with pernicious anemia have concomitant iron deficiency anemia and, in severe cases, may have microcytic red blood cells ⁶⁷. Serum levels of iron, transferrin, and ferritin should be measured in patients with pernicious anemia, especially when macrocytosis is not present. It is important to remember that patients with pernicious anemia and associated megaloblastic anemia may also develop severe thrombocytopenia ⁸⁹. Platelet production can be reduced by around 10%, and patients may also have abnormal platelet function ⁸⁹. Leukopenia may be present but rarely causes any clinical issues ⁸⁹. Indirect bilirubin and lactate dehydrogenase levels are usually elevated due to the rapid breakdown of red blood cells and intramedullary hemolysis. It is imperative to remember that vitamin B12 and folate levels should be tested simultaneously in patients with macrocytic anemia to ensure both deficiencies are diagnosed if present ²³.

Pernicious anemia treatment

Doctors treat pernicious anemia by replacing the missing vitamin B12 in the body. People who have pernicious anemia may need lifelong treatment ²³. Parenteral administration is the preferred method of administration and should begin with a 1000 mcg of either cyanocobalamin or hydroxocobalamin intramuscular (IM) injection every day or every other day for the first week ⁵⁴, ¹⁰⁸. This is followed by weekly injections for 1 to 2 months, and then maintenance therapy with monthly injections is provided ⁵⁴. An alternate approach is an initial dose of 1000 mcg IM injection every other day for 1 to 2 weeks, followed by weekly injections for one month and then monthly injections thereafter ⁸⁹. Response should be monitored by reticulocyte counts, lactic dehydrogenase (LDH), and an appropriate rise in hemoglobin levels. LDH levels decrease and hemoglobin levels increase by about 1 g/dL/week. A rise in LDH might indicate a relapse.

Recent data have suggested that high-dose oral replacement is as effective as IM replacement because passive absorption of vitamin B12 can occur in the absence of intrinsic factor (IF) ¹⁰⁹. Even with a total absence of intrinsic factor (IF), about 1% of an oral dose is absorbed, and the daily requirement for vitamin B12 is 1 µg/day. A study by Zhang and colleagues ¹¹⁰ found evidence that using orally ingested soy protein isolate (SPI) nanoparticles as a carrier can improve the intestinal transport and absorption of vitamin B12. A 2018 Cochrane review reported oral route was as effective as IM vitamin B12 replacement; however, this was supported by very low-quality evidence ²⁵. Therefore, current guidelines advise against the use of oral replacement in the initial phase of treatment ²³. High-dose oral replacement (with 1000 to 2000 mcg of vitamin B12 daily) can be considered in patients with pernicious anemia for maintenance therapy ²³.

The oral route may be necessary in the rare patients who have allergic reactions to parenteral administration, or in patients receiving anticoagulant or antiplatelet agent therapy, in whom intramuscular injections are contraindicated ¹¹¹. If the oral route is used, obtain serum cobalamin measurements at periodic intervals to ensure that adequate quantities of cobalamin have been absorbed. Oral cobalamin therapy should not be used in patients with neurologic symptoms ²³.

A randomized, placebo-controlled trial of oral cobalamin therapy in 50 patients with borderline serum vitamin B12 levels (125-200 pg/mL) and nonspecific symptoms compatible with subtle vitamin B12 deficiency found that after 1 month, serum methylmalonic acid (MMA) levels were corrected more often in patients receiving oral cobalamin than in those receiving placebo. However, the benefit to the MMA level disappeared after 3 additional months without cobalamin therapy ¹¹².

A study found that oral cobalamine was more effective than parenteral therapy in some circumstances ¹¹².

The earliest sign of treatment response is an increase in reticulocyte count that occurs within 5 to 14 days of treatment ⁶⁷. A decrease in methylmalonic acid (MMA) and plasma homocysteine levels has also been observed in the first five days of treatment. Sustained normalization of serum cobalamin subsequently follows. Neuropsychiatric symptoms take a longer time to recover. As noted before, some neurologic symptoms may be irreversible.

Alternate formulations of vitamin B12 have been approved and include sublingual and intranasal formulations. However, data regarding their clinical efficacy is still limited, and they are not routinely recommended.

Patients whose vitamin B12 deficiency is due to underlying diseases involving the intestine or pancreas may require additional therapy ¹¹³. Examples of additional therapy are surgical correction of anatomic abnormalities of the gut that produce small bowel bacterial overgrowth, or the treatment of fish tapeworm anemia or pancreatitis. Elderly patients who also have hypokalemia should receive oral potassium supplements, to prevent severe hypokalemia and possible arrhythmias.

Blood transfusions are rarely required in patients with a megaloblastic anemia that is due to vitamin B12 deficiency ¹¹⁴. The likelihood of obtaining a dramatic response to cobalamin therapy within a few days of initiating treatment makes it unnecessary to subject the patient to the hazards of blood transfusion. Usually, mild-to-moderate congestive heart failure secondary to anemia abates with bed rest and low-dosage diuretic therapy. However, if the congestive heart failure is severe or the patient has coronary insufficiency, transfusion of packed red blood cells may be necessary ¹¹⁴. Transfuse the blood slowly because patients who are transfused for severe anemia often develop circulatory overload. For this reason, low-dose diuretic therapy is often employed with the blood transfusion ¹¹⁴.

Prognosis for pernicious anemia

Most people with pernicious anemia do well with treatment ⁸⁸. It is important to start treatment early. Nerve damage can be permanent if treatment does not start within 6 months of symptoms. A 2006 observational study evaluating 57 patients with subacute combined degeneration reported only 14% clinical resolution after B12 treatment ¹¹⁵. Still, the study reported that of these patients, 86% had at least some clinical improvement. Subgroup analysis revealed that the absence of sensory dermatomal deficit, negative Romberg and Babinski signs, age less than 50 years, and less than or equal to 7-segment involvement on magnetic resonance imaging correlated with complete resolution of neurologic symptoms ¹¹⁵. This study highlights the importance of early diagnosis and treatment, as patients with severe or prolonged neurological symptoms tend to have persistent symptoms despite treatment.

With ongoing care and proper treatment, most people who have pernicious anemia can recover, feel well, and live normal lives.

Without treatment, pernicious anemia can lead to serious problems with the heart, nerves, and other parts of the body. Some of these problems may be permanent.

Although vitamin B12 therapy resolves the anemia, it will not cure atrophic gastritis, and patients with pernicious anemia remain at higher risk of gastric cancers throughout their life ⁹⁶. There is an increased risk of gastric adenocarcinoma in these patients ⁹⁸. A longitudinal study from China reported that patients with pernicious anemia and anti-IF or anti-parietal cell antibodies had an unsatisfactory neurologic response to treatment and, over time, developed newly diagnosed hypothyroidism as well as alimentary canal malignancies ¹¹⁶. Twenty percent of the cancers noted in this study were gastric cancers. This study reported a mean survival of 64 months for patients who developed a malignancy compared to 129 months for those without malignancy. The mortality rate was reported as 31%, with cancer-related deaths representing 37%. Interestingly, the authors reported a higher risk of cancer in patients who were positive for anti-IF antibodies compared to those without these antibodies ¹¹⁶.

Vitamin B12 deficiency causes

Vitamin B-12 deficiency can result from:

• Inadequate intake
• Inadequate absorption
• Decreased utilization
• Use of certain drugs

Intestinal malabsorption, rather than inadequate dietary intake, can explain most cases of vitamin B12 deficiency ¹¹⁷. Absorption of vitamin B12 from food requires normal function of the stomach, pancreas, and small intestine. Stomach acid and enzymes free vitamin B12 from food, allowing it to bind to R-protein (also known as transcobalamin-1 or haptocorrin), found in saliva and gastric fluids. In the alkaline environment of the small intestine, R-proteins are degraded by pancreatic enzymes, freeing vitamin B12 to bind to intrinsic factor (IF), a protein secreted by specialized cells in the stomach. Receptors on the surface of the ileum (final part of the small intestine) take up the IF-B12 complex only in the presence of calcium, which is supplied by the pancreas ¹¹⁷.

Vitamin B12 can also be absorbed by passive diffusion, but this process is very inefficient—only about 1% absorption of the vitamin B12 dose is absorbed passively ¹¹⁸. The prevalent causes of vitamin B12 deficiency are (1) an autoimmune condition known as pernicious anemia, and (2) a disorder called food-bound vitamin B12 malabsorption. Both conditions have been associated with a chronic inflammatory disease of the stomach known as atrophic gastritis.

In the elderly, inadequate absorption most commonly results from decreased acid secretion. In such cases, crystalline Vitamin B-12 (such as that available in vitamin supplements) can be absorbed, but food-bound vitamin B-12 is not liberated and absorbed normally.

Inadequate absorption may occur in blind loop syndrome (with overgrowth of bacteria) or fish tapeworm infestation; in these cases, bacteria or parasites use ingested Vitamin B-12 so that less is available for absorption.

Vitamin B-12 absorption may be inadequate if ileal absorptive sites are destroyed by inflammatory bowel disease or are surgically removed.

Less common causes of inadequate vitamin B-12 absorption include chronic pancreatitis, gastric or bariatric surgery, malabsorption syndromes, AIDS, use of certain drugs (eg, antacids, metformin), repeated exposure to nitrous oxide, and a genetic disorder causing malabsorption in the ileum (Imerslund-Graesbeck syndrome).

Less commonly, decreased utilization of Vitamin B-12 or use of certain drugs causes Vitamin B-12 deficiency.

Atrophic gastritis

Atrophic gastritis is a histopathologic entity characterized by chronic inflammation of the gastric mucosa with loss of the gastric glandular cells and replacement by intestinal-type epithelium, pyloric-type glands, and fibrous tissue as a response to chronic injury ¹¹⁹. Atrophy of the gastric mucosa is the endpoint of chronic processes, such as chronic gastritis associated with Helicobacter pylori infection, other unidentified environmental factors, and autoimmunity directed against gastric glandular cells (autoimmune gastritis) ¹¹⁹. Atrophic gastritis represents the end stage of chronic gastritis, both infectious and autoimmune. In both cases, the clinical manifestations of atrophic gastritis are those of chronic gastritis, but pernicious anemia is observed specifically in patients with autoimmune gastritis and not in those with Helicobacter pylori–associated atrophic gastritis.

Atrophic gastritis is thought to affect 10%-30% of people over 60 years of age ¹²⁰. Atrophic gastritis is frequently associated with the presence of autoantibodies directed toward stomach cells (see pernicious anemia) and/or infection by the bacteria, Helicobacter pylori (H. pylori) ¹²¹. Helicobacter pylori (H. pylori) infection induces chronic inflammation of the stomach, which may progress to peptic ulcer disease, atrophic gastritis, and/or gastric cancer in some individuals ¹²². Diminished gastric function in individuals with atrophic gastritis can result in bacterial overgrowth in the small intestine and cause food-bound vitamin B12 malabsorption. Vitamin B12 levels in serum, plasma, and gastric fluids are significantly decreased in individuals with H. pylori infection, and eradication of the bacteria has been shown to significantly improve vitamin B12 serum concentrations ¹²³.

Pernicious anemia

Pernicious anemia has been estimated to be present in approximately 2% of individuals over 60 years of age ¹²⁴. Although anemia is often a symptom, pernicious anemia is actually the end stage of an autoimmune inflammation of the stomach known as autoimmune atrophic gastritis, resulting in destruction of stomach cells by one’s own antibodies (autoantibodies). Progressive destruction of the cells that line the stomach causes decreased secretion of acid and enzymes required to release food-bound vitamin B12. Antibodies to intrinsic factor (IF) bind to IF preventing formation of the IF-B12 complex, further inhibiting vitamin B12 absorption. About 20% of the relatives of pernicious anemia patients also have the condition, suggesting a genetic predisposition. It is also thought that H. pylori infection could be involved in initiating the autoimmune response in a subset of individuals ¹²⁵. Furthermore, co-occurrence of autoimmune atrophic gastritis with other autoimmune conditions, especially autoimmune thyroiditis and type 1 diabetes mellitus, has been reported ¹²⁶, ¹²⁷.

Treatment of pernicious anemia generally requires injections of vitamin B12 to bypass intestinal absorption. High-dose oral supplementation is another treatment option, because consuming 1,000 μg (1 mg)/day of vitamin B12 orally should result in the absorption of about 10 μg/day (1% of dose) by passive diffusion. In fact, high-dose oral therapy is considered to be as effective as intramuscular injection ¹.

Food-bound vitamin B12 malabsorption

Food-bound vitamin B12 malabsorption is defined as an impaired ability to absorb food- or protein-bound vitamin B12; individuals with this condition can fully absorb the free form ¹²⁸. While the condition is the major cause of poor vitamin B12 status in the elderly population, it is usually associated with atrophic gastritis, a chronic inflammation of the lining of the stomach that ultimately results in the loss of glands in the stomach (atrophy) and decreased stomach acid production (see atrophic gastritis). Because stomach acid is required for the release of vitamin B12 from the proteins in food, vitamin B12 absorption is diminished. Decreased stomach acid production also provides an environment conducive to the overgrowth of anaerobic bacteria in the stomach, which further interferes with vitamin B12 absorption ⁴¹. Because vitamin B12 in supplements is not bound to protein, and because intrinsic factor (IF) is still available, the absorption of supplemental vitamin B12 is not reduced as it is in pernicious anemia. Thus, individuals with food-bound vitamin B12 malabsorption do not have an increased requirement for vitamin B12; they simply need it in the crystalline form found in fortified foods and dietary supplements.

Inherited disorders of vitamin B12 absorption

Rare cases of inborn errors of vitamin B12 metabolism have been reported in the literature ¹¹⁷. Imerslund-Gräsbeck syndrome is an inherited vitamin B12 malabsorption syndrome that causes megaloblastic anemia and neurologic disorders of variable severity in affected subjects. Similar clinical symptoms are found in individuals with hereditary IF deficiency (also called congenital pernicious anemia) in whom the lack of IF results in the defective absorption of vitamin B12. Additionally, mutations affecting vitamin B12 transport in the body have been identified ¹²⁹.

Other causes of vitamin B12 deficiency

Other causes of vitamin B12 deficiency include surgical resection of the stomach or portions of the small intestine where receptors for the IF-B12 complex are located. Conditions affecting the small intestine, such as malabsorption syndromes (celiac disease and tropical sprue), may also result in vitamin B12 deficiency. Because the pancreas provides critical enzymes, as well as calcium required for vitamin B12 absorption, pancreatic insufficiency may contribute to vitamin B12 deficiency. Since vitamin B12 is found only in foods of animal origin, a strict vegetarian (vegan) diet has resulted in cases of vitamin B12 deficiency. Moreover, alcoholics may experience reduced intestinal absorption of vitamin B12 ¹¹⁸ and individuals with acquired immunodeficiency syndrome (AIDS) appear to be at increased risk of deficiency, possibly related to a failure of the IF-B12 receptor to take up the IF-B12 complex ⁴¹. Furthermore, long-term use of acid-reducing drugs has also been implicated in vitamin B12 deficiency (see drug interactions below).

Drug interactions

A number of drugs reduce the absorption of vitamin B12. Proton-pump inhibitors (e.g., omeprazole and lansoprazole), used for therapy of Zollinger-Ellison syndrome and gastroesophageal reflux disease (GERD), markedly decrease stomach acid secretion required for the release of vitamin B12 from food but not from supplements. Long-term use of proton-pump inhibitors has been found to decrease blood vitamin B12 levels. However, vitamin B12 deficiency does not generally develop until after at least three years of continuous therapy ¹³⁰, ¹³¹. Another class of gastric acid inhibitors known as histamine-2 (H2)-receptor antagonists (e.g., cimetidine, famotidine, and ranitidine), often used to treat peptic ulcer disease, has also been found to decrease the absorption of vitamin B12 from food. It is not clear whether the long-term use of H2-receptor antagonists could cause overt vitamin B12 deficiency ¹³², ¹³³. Individuals taking drugs that inhibit gastric acid secretion should consider taking vitamin B12 in the form of a supplement because gastric acid is not required for its absorption. Other drugs found to inhibit vitamin B12 absorption from food include cholestyramine (a bile acid-binding resin used in the treatment of high cholesterol), chloramphenicol and neomycin (antibiotics), and colchicine (medicine for gout treatment). Metformin, a medication for individuals with type 2 diabetes, was found to decrease vitamin B12 absorption by tying up free calcium required for absorption of the IF-B12 complex ¹³⁴. However, the clinical significance of this is unclear ¹³⁵. It is not known whether calcium supplementation can reverse vitamin B12 malabsorption; therefore, calcium supplementation is not currently prescribed for the prevention or treatment of metformin-induced vitamin B12 deficiency ¹³⁶. Previous reports that megadoses of vitamin C destroy vitamin B12 have not been supported ¹³⁷ and may have been an artifact of the assay used to measure vitamin B12 levels ¹³⁸.

Nitrous oxide, a commonly used anesthetic, oxidizes and inactivates vitamin B12, thus inhibiting both of the vitamin B12-dependent enzymes, and can produce many of the clinical features of vitamin B12 deficiency, such as megaloblastic anemia or neuropathy. Since nitrous oxide is commonly used for surgery in the elderly, some experts feel vitamin B12 deficiency should be ruled out prior to its use ¹²⁰, ¹³⁹.

Large doses of folic acid given to an individual with an undiagnosed vitamin B12 deficiency could correct megaloblastic anemia without correcting the underlying vitamin B12 deficiency, leaving the individual at risk of developing irreversible neurologic damage ¹³⁸. For this reason, the Food and Nutrition Board of the US Institute of Medicine advises that all adults limit their intake of folic acid (supplements and fortification) to 1,000 μg (1 mg) daily ¹³⁸.

Vitamin B12 deficiency prevention

Because of potential interactions from prolonged medication use, physicians should consider screening patients for vitamin B12 deficiency if they have been taking proton pump inhibitors or H2 blockers for more than 12 months, or metformin for more than four months. The average intake of vitamin B12 in the United States is 3.4 mcg per day, and the recommended dietary allowance is 2.4 mcg per day for adult men and nonpregnant women, and 2.6 mcg per day for pregnant women.30 Patients older than 50 years may not be able to adequately absorb dietary vitamin B12 and should consume food fortified with vitamin B12 ²⁹. Vegans and strict vegetarians should be counseled to consume fortified cereals or supplements to prevent deficiency. The American Society for Metabolic and Bariatric Surgery recommends that patients who have had bariatric surgery take 1 mg of oral vitamin B12 per day indefinitely ¹⁴⁰.

Vitamin B12 deficiency signs and symptoms

Vitamin B12 deficiency results in impairment of the activities of vitamin B12-requiring enzymes. Impaired activity of methionine synthase results in elevated homocysteine levels, while impaired activity of L-methylmalonyl-CoA mutase results in increased levels of a metabolite of methylmalonyl-CoA called methylmalonic acid (MMA). Vitamin B12 deficiency affects multiple systems, and consequences vary in severity from mild fatigue to severe neurologic impairment ¹⁴¹, ⁴, ⁷.

Individuals with mild vitamin B12 deficiency may not experience symptoms, but their blood levels of homocysteine and/or MMA (methylmalonic acid) may be elevated ¹³⁹.

Vitamin B12 deficiency is generally characterized by a specific type of anemia called megaloblastic anemia. Anemia usually develops insidiously. It can cause fatigue (easily fatigued with exertion), palpitations, pale skin, weakness, constipation, loss of appetite, and weight loss ⁴, ⁷. Megaloblastic anemia is often more severe than its symptoms indicate because its slow evolution allowing physiologic adaptation.

Subacute combined degeneration refers to degenerative changes to the various parts of the spinal cord, including the dorsal columns, the lateral corticospinal tracts, and the spinocerebellar tracts due to vitamin B-12 deficiency; they affect mostly brain and spinal cord white matter ¹⁴². Demyelinating or axonal peripheral neuropathies can occur ¹⁴³. In early stages, decreased position (proprioception) and vibratory sensation in the extremities is accompanied by mild to moderate weakness and hyporeflexia. In later stages, spasticity, extensor plantar responses, greater loss of position and vibratory sensation in the lower extremities, and ataxia emerge ¹⁴⁴, ²⁸. These deficits may develop in a stocking-glove distribution. Tactile, pain, and temperature sensations are usually spared but may be difficult to assess in the elderly. Areflexia can be permanent if neuronal death occurs in the posterior and lateral spinal cord tracts ¹⁴⁵, ¹⁹.

Some patients are also irritable and mildly depressed. Dementia-like disease, including episodes of psychosis, paranoia (megaloblastic madness), poor memory, delirium, depression, confusion, and, at times, postural hypotension may occur in advanced cases ¹⁴⁵, ⁴. The confusion may be difficult to differentiate from age-related dementias, such as Alzheimer disease.

Neurologic symptoms may develop independently from and often without hematologic abnormalities. Clinical evaluation seems to show an inverse relationship between the severity of megaloblastic anemia and the degree of neurologic impairment ⁷.

Occasionally, splenomegaly and hepatomegaly occur. Various gastrointestinal symptoms, including weight loss and poorly localized abdominal pain, may occur. Glossitis, usually described as burning of the tongue, is uncommon.

Symptoms of B12 deficiency can take decades to develop, and can usually only be diagnosed by a medical professional ⁴, ⁵.

General symptoms of anemia may include:

• extreme tiredness (fatigue)
• lack of energy (lethargy)
• breathlessness
• feeling faint
• headaches
• pale skin
• noticeable heartbeats (palpitations)
• hearing sounds coming from inside the body, rather than from an outside source (tinnitus)
• loss of appetite and weight loss

If you have anemia caused by a vitamin B12 deficiency, you may have other symptoms, such as ¹⁵, ¹⁴¹:

• a pale yellow tinge to your skin. In advanced anemia, severe pale skin with jaundice (due to hemolysis) produces a “peculiar lemon-yellow” skin color ⁸⁹.
• vitiligo
• skin hyperpigmentation
• a sore and red tongue (glossitis)
• mouth ulcers
• pins and needles (paresthesia)
• changes in the way that you walk and move around (gait abnormalities)
• disturbed vision
• irritability
• depression
• changes in the way you think, feel and behave
• a decline in your mental abilities, such as memory, understanding and judgement (dementia)
• acute psychosis
• areflexia
• loss of proprioception and vibratory sense
• impaired sense of smell

Some of these symptoms can also happen in people who have a vitamin B12 deficiency but have not developed anemia.

Megaloblastic anemia

Diminished activity of methionine synthase in vitamin B12 deficiency inhibits the regeneration of tetrahydrofolate (THF) and traps folate in a form that is not usable by the body, resulting in symptoms of folate deficiency even in the presence of adequate folate levels. Thus, in both folate and vitamin B12 deficiencies, folate is unavailable to participate in DNA synthesis. This impairment of DNA synthesis affects the rapidly dividing cells of the bone marrow earlier than other cells, resulting in the production of large, immature, hemoglobin-poor red blood cells. The resulting anemia is known as megaloblastic anemia and is the symptom for which the disease, pernicious anemia, was named ⁴¹. Supplementation with folic acid will provide enough usable folate to restore normal red blood cell formation. However, if vitamin B12 deficiency is the cause, it will persist despite the resolution of the anemia. Thus, megaloblastic anemia should not be treated with folic acid until the underlying cause has been determined ¹⁴⁶.

Neurologic symptoms

The neurologic symptoms of vitamin B12 deficiency include numbness and tingling of the hands and, more commonly, the feet; difficulty walking; memory loss; disorientation; and dementia with or without mood changes. Although the progression of neurologic complications is generally gradual, such symptoms may not be reversed with treatment of vitamin B12 deficiency, especially if they have been present for a long time. Neurologic complications are not always associated with megaloblastic anemia and are the only clinical symptom of vitamin B12 deficiency in about 25% of cases ²⁹. Although vitamin B12 deficiency is known to damage the myelin sheath covering cranial, spinal, and peripheral nerves, the biochemical processes leading to neurological damage in vitamin B12 deficiency are not yet fully understood ¹⁴⁷.

Gastrointestinal symptoms

Tongue soreness, appetite loss, and constipation have also been associated with vitamin B12 deficiency. The origins of these symptoms are unclear, but they may be related to the stomach inflammation underlying some cases of vitamin B12 deficiency and to the progressive destruction of the lining of the stomach ²⁹.

Hyperhomocysteinemia

High levels of homocysteine in the blood (hyperhomocysteinemia) has been linked to heart disease and stroke ². Hyperhomocysteinemia can be caused by a deficiency of either vitamin B12 or folate, and in human subjects mild (13–24 µM) and moderate (25–60 µM) hyperhomocysteinemia are also associated with mutations of MTHFR genes.

Vitamin B12 deficiency hyperhomocysteinemia may be associated with osteoporosis, depression, cognitive decline, and some forms of dementia in the elderly ². More recently, vitamin B12 deficiency has been reported as common among patients with hyperhomocysteinemia and thrombosis ¹⁴⁸, although the presence of a direct effect of vitamin B12 deficiency rather than mediated by hyperhomocysteinemia or other factors is uncertain. In fact, lifestyle-related factors, such as smoking status, body mass index (BMI), and physical activity, could interfere between hyperhomocysteinemia and the thromboembolism relationship ¹⁴⁹. Moreover, the effect of lowering homocysteine levels in patients with intermediate (total homocysteine 30–100 µmol/L) or severe hyperhomocysteinemia (total homocysteine > 100 µmol/L) remains unknown ¹⁵⁰. The cases described below report examples of vitamin B12 deficiency and hyperhomocysteinemia related to different causes.

A case of cerebral venous thrombosis secondary to hyperhomocysteinemia caused by vitamin B12 deficiency in a 32-year-old Indo-Aryan man who followed a strict vegetarian diet is reported by Kapur ¹⁵¹. The preliminary blood examination revealed macrocytic anemia with hemoglobin of 11.4 g/dL and mean corpuscular volume (MCV) of 110 fL ¹⁵¹. Peripheral blood film showed macrocytes and macro-ovalocytes with hypersegmented neutrophils; low serum cobalamin levels 68 pg/mL (200–600) with normal folate levels and total serum homocysteine
levels of 36 μmol/L (5.0–13.9) were observed ¹⁵¹. In addition to other treatments, the patient received parenteral cyanocobalamin 1000 μg once daily for seven days. Gradually, he regained sensorium, his power improved, and he was discharged on orally administered sodium valproate, warfarin, and methylcobalamin. Repeated investigations undertaken at six months after stopping anticoagulants showed normal serum cobalamin 364 pg/mL (200–600) and fasting total homocysteine levels 8.4 μmol/L. The authors conclude that hyperhomocysteinemia is an independent risk factor for cerebral venous thrombosis in patients with cobalamin deficiency, especially those who follow a strict vegetarian diet, and that hyperhomocysteinemia can be easily reversed with vitamin supplementation, cobalamin, and folic acid ¹⁵¹.

The cases of four Moroccan patients with acute vein thrombosis of different sites are reported by Ammouri ¹⁵². Three men and one woman of different ages (a 34-year-old man, a 60-year-old man, a 58-year-old man, and a 47-year-old woman) were selected. All patients presented low hemoglobin level (from 8.6 g/dL to 9.5 g/dL), low MCV (mean corpuscular volume), low cobalamin plasma level (about 60 pg/mL; normal >120 pg/mL), and high levels of plasma homocysteine (50 to 200 μmol/L; normal range <15 µmol/L) with normal folate plasma levels. For all, pernicious anemia and venous thrombosis secondary to hyperhomocysteinemia were evident. First, the authors speculated that normal folate levels may have contributed to the delay in the diagnosis of pernicious anemia, leading to severe hyperhomocysteinemia and the consequent development of vascular injury ¹⁵².

Hyperhomocysteinemia could lead to venous thrombosis by several pathways. For example, the toxic effect of homocysteine on the vascular endothelium and on the dotting cascade, as well procoagulant properties of homocysteine, including the decrease of antithrombin III binding to endothelial heparan sulfate, an increase of affinity between lipoprotein(a) and fibrin, induction of tissue factor activity in endothelial cells, and inhibition of inactivation of factor V by activated protein. In all patients, clinical and biological abnormalities disappeared upon vitamin B12 supplementation. The authors concluded that vitamin B12 supplements can rapidly correct hyperhomocysteinemia avoiding and preventing thrombotic events ¹⁵².

Tanaka et al. ¹⁵⁰ reported a case of a 39-year-old man with inferior vena cava (IVC) thrombus. The analysis of risk factors of venous thromboembolism shown hyperhomocysteinemia (total homocysteine 83.1 µmol/L; normal range 5–15 µmol/L) due to an unbalanced diet with a deficiency of folic acid and vitamin B12. The patient was treated with both folic acid and vitamin B6/vitamin B12 supplementation in association with warfarin, inducing a significant resolution of thrombus after four weeks and no evidence of recurrent IVC thrombus at six months. The authors concluded that B vitamins and folic acid therapy might be effective in patients with severe hyperhomocysteinemia ¹⁵⁰.

An interesting case of a 43-year-old man presenting with a two-week history of painless ascending sensory disturbances, suspected to be suffering from acute inflammatory polyneuropathy, is reported by Ulrich et al. ¹⁵³. On clinical examination, deep tendon reflexes were preserved, muscle strength was 5/5 everywhere, and gait was ataxic. Initial laboratory assessment showed nearly normal holotranscobalamin (43 pmol/L; pmol/L normal >50 pmol/L), suggesting no vitamin B12 deficiency. Surprisingly, further investigation showed high homocysteine (48.5 µmol/L; normal <10 µmol/L), suggesting an impairment of vitamin B12-dependent metabolism leading to the diagnosis of subacute combined degeneration. The patient remembered having taken tablets containing cobalamin for three days before hospitalization. The authors concluded that holotranscobalamin can be rapidly normalized during supplementation and the analysis of methylmalonic acid (MMA) and homocysteine might help to detect B12 deficiency in patients who recently started supplementation.

A case of a 24-year-old male with unprovoked bilateral submassive pulmonary emboli with a high level of homocysteine without anemia is reported by Kovalenko et al. ¹⁵⁴. Complete blood count showed a MCV of 104fL without anemia, and homocysteine level was 41.3 μmol/L (normal 4.0–13.7 μmol/L). Workup for macrocytosis was notable for low vitamin B12 (72 pg/mL) and folate (2.1 ng/mL) levels. After vitamin B12 supplementation, serum homocysteine levels did not decrease to normal values. The authors speculated that a poor absorption of B vitamins due to a small bowel resection two years before and excessive alcohol consumption could have impaired the results. Another case associated with alcoholism was previously described by Goette et al. ¹⁵⁵. The authors described a rare case of a 32-year-old man with severe hyperhomocysteinemia underlying a probable cause of thromboembolic complications ¹⁵⁵. The patient did not have a history of cardiovascular disease, but he had at least a six-month history of alcohol abuse at least six months before hospital admission. Laboratory assays showed abnormalities in liver functions, vitamin B12 (226 pg/mL; normal range 150–675 pg/mL) and folate (1.6 μg/L; normal range 1.4–11.8 μg/L) were low but within normal range, while serum homocysteine was at least 12 times higher than normal (173 μmol/L). The patient was treated with 5 mg oral folic acid and 20 mg oral vitamin B6 daily. Vitamin supplementation was then adapted and integrated with other drugs, such as weight-adapted low molecular weight heparin and L-arginine. For some patients, the authors suggested the screening for hyperhomocysteinemia in association with endothelial dysfunction markers as appropriate ¹⁵⁵.

Elevated plasma homocysteine is involved in cognitive decline, including Alzheimer’s disease, mild cognitive impairment, and dementia, especially in elderly subjects. McCaddon ¹⁵⁶ reported seven cases of older patients (four women aged 78 years, 84 years, 77 years and 87 years, 84 years old, and two men 71 and 75 years old). They presented with cognitive impairment and/or depression and dementia ¹⁵⁶. Each had different vitamin B12 status with hyperhomocysteinemia. Treatment with N-acetylcysteine, together with B vitamin supplements, improves cognitive status in hyperhomocysteinemic patients. The authors concluded that it could be important to evaluate inadequate vitamin B12 and folate metabolism in subjects with cognitive diseases, underlining the importance of clinical trials to evaluate the beneficial effects of a synergistic approach to cognitively impaired hyperhomocysteinaemic patients ¹⁵⁶.

Complications of vitamin B12 deficiency

A lack of vitamin B12 (with or without anemia) can cause complications.

Vitamin B12 deficiency complications can include ¹⁵⁷:

• Heart failure due to the anemia
• Severe disabling neurological deficits
• Risk of gastric cancer
• Risk of developing an autoimmune disorder like type 1 diabetes, myasthenia gravis, Hashimoto disease, or rheumatoid arthritis

Neurological problems

A lack of vitamin B12 can cause neurological problems, which affect your nervous system, such as:

• vision problems
• memory loss
• pins and needles (paraesthesia)
• loss of physical co-ordination (ataxia), which can affect your whole body and cause difficulty speaking or walking
• damage to parts of the nervous system (peripheral neuropathy), particularly in the legs

If neurological problems do develop, they may be irreversible.

Stomach cancer

If you have a vitamin B12 deficiency caused by pernicious anemia, a condition where your immune system attacks healthy cells in your stomach, your risk of developing stomach cancer is increased.

Neural tube defects

If you’re pregnant, not having enough vitamin B12 can increase the risk of your baby developing a serious birth defect known as a neural tube defect. The neural tube is a narrow channel that eventually forms the brain and spinal cord.

Examples of neural tube defects include:

• Spina bifida – where the baby’s spine does not develop properly
• Anencephaly – where a baby is born without parts of the brain and skull
• Encephalocele – where a membrane or skin-covered sac containing part of the brain pushes out of a hole in the skull

Infertility

Vitamin B12 deficiency can sometimes lead to temporary infertility, an inability to conceive. Infertility usually improves with appropriate vitamin B12 treatment.

Anemia complications

All types of anemia, regardless of the cause, can lead to heart and lung complications as the heart struggles to pump oxygen to the vital organs.

Adults with severe anemia are at risk of developing:

• an abnormally fast heartbeat (tachycardia)
• heart failure, where the heart fails to pump enough blood around the body at the right pressure

Vitamin B12 deficiency diagnosis

• Complete blood test checking for anemia and vitamin B-12 and folate levels
• Sometimes methylmalonic acid levels or Schilling test

It is important to remember that severe neurologic disease may occur without anemia or macrocytosis.

Figure 6. Vitamin B12 deficiency diagnosis

Footnote: Suggested approach to the patient with suspected vitamin B12 deficiency.

[Source ⁵ ]

Table 5 lists the relative sensitivities and specificities of various laboratory tests ¹⁵⁸

Table 5. Vitamin B12 deficiency laboratory tests sensitivities and specificities

[Source ⁵ ]

Vitamin B12 deficiency test

Diagnosis of Vitamin B-12 deficiency is based on complete blood count and Vitamin B-12 and folate levels. Complete blood count usually detects megaloblastic anemia. Tissue deficiency and macrocytic indexes may precede the development of anemia. A Vitamin B-12 level < 200 pg/mL (< 145 pmol/L) indicates Vitamin B-12 deficiency. The folate level is measured because Vitamin B-12 deficiency must be differentiated from folate deficiency as a cause of megaloblastic anemia; folate supplementation can mask Vitamin B-12 deficiency and may alleviate megaloblastic anemia but allow the neurologic deficits to progress or even accelerate.

When clinical judgment suggests Vitamin B-12 deficiency but the Vitamin B-12 level is low-normal (200 to 350 pg/mL [145 to 260 pmol/L]) or hematologic indexes are normal, other tests can be done. They include measuring the following:

• Serum methylmalonic acid (MMA) levels: An elevated MMA level supports Vitamin B-12 deficiency but may be due to renal failure. Methylmalonic acid (MMA) levels can also be used to monitor the response to treatment. Methylmalonic acid levels remain normal in folate deficiency.
• Homocysteine levels: Levels may be elevated with either Vitamin B-12 or folate deficiency.
• Less commonly, holotranscobalamin II (transcobalamin II–B12 complex) content: When holotranscobalamin II is < 40 pg/mL (< 30 pmol/L), Vitamin B-12 is deficient.

After Vitamin B-12 deficiency is diagnosed, additional tests (eg, Schilling test) may be indicated for younger adults but usually not for the elderly. Unless dietary Vitamin B-12 is obviously inadequate, serum gastrin levels or autoantibodies to intrinsic factor may be measured; sensitivity and specificity of these tests may be poor.

Schilling test

The Schilling test is useful only if diagnosing intrinsic factor deficiency is important, as in classic pernicious anemia. This test is not necessary for most elderly patients. The Schilling test measures absorption of free radiolabeled Vitamin B-12. Radiolabeled Vitamin B-12 is given orally, followed in 1 to 6 h by 1000 mcg (1 mg) of parenteral Vitamin B-12, which reduces uptake of radiolabeled Vitamin B-12 by the liver. Absorbed radiolabeled Vitamin B-12 is excreted in urine, which is collected for 24 h. The amount excreted is measured, and the percentage of total radiolabeled Vitamin B-12 is determined. If absorption is normal, ≥ 9% of the dose given appears in the urine. Reduced urinary excretion (< 5% if kidney function is normal) indicates inadequate Vitamin B-12 absorption. Improved absorption with the subsequent addition of intrinsic factor to radiolabeled Vitamin B-12 confirms the diagnosis of pernicious anemia.

The test is often difficult to do or interpret because of incomplete urine collection or renal insufficiency. In addition, because the Schilling test does not measure absorption of protein-bound Vitamin B-12, the test does not detect defective liberation of Vitamin B-12 from foods, which is common among the elderly. The Schilling test repletes Vitamin B-12 and can mask deficiency, so it should be done only after all other diagnostic tests and therapeutic trials.

If malabsorption is identified, the Schilling test can be repeated after a 2-week trial of an oral antibiotic. If antibiotic therapy corrects malabsorption, the likely cause is intestinal overgrowth of bacteria (eg, blind-loop syndrome).

Vitamin B12 deficiency treatment

Vitamin B12 deficiency can be treated with intramuscular injections of cyanocobalamin or hydroxocobalamin or oral vitamin B12 therapy. However, depending on the cause of the B12 deficiency, the duration and route of treatment vary. In patients who are B12 deficient due to a strict vegan diet, an oral supplement of B12 is adequate for repletion ²⁴. A 2018 Cochrane review included three randomized controlled trials (RCTs) that compared very high doses (1,000–2,000 mcg) of oral with intramuscular vitamin B12 for vitamin B12 deficiency in a total of 153 participants ²⁵. The evidence from these studies, although of low quality, showed that the ability of high oral doses of vitamin B12 supplements to normalize serum vitamin B12 was similar to that of intramuscular vitamin B12. The British Society for Haematology recommends intramuscular vitamin B12 for severe deficiency and malabsorption syndromes, whereas oral replacement may be considered for patients with asymptomatic, mild disease with no absorption or compliance concerns ²³.

There are 2 types of vitamin B12 injections:

• Hydroxocobalamin
• Cyanocobalamin

Typically, vitamin B12 deficiency is treated with intramuscular injections of cyanocobalamin or hydroxocobalamin, because this method bypasses any barriers to absorption. Hydroxocobalamin is usually the recommended option as it stays in the body for longer. Approximately 10% of the standard injectable dose of 1 mg is absorbed, which allows for rapid replacement in patients with severe deficiency or severe neurologic symptoms ⁷. Guidelines from the British Society for Haematology recommend injections three times per week for two weeks in patients without neurologic deficits ²³. If neurologic deficits are present, injections should be given every other day for up to three weeks or until no further improvement is noted.

If vitamin B12 deficiency coexists with folate deficiency, vitamin B12 should be replaced first to prevent subacute combined degeneration of the spinal cord ⁴.

Although hematologic abnormalities are usually corrected within 6 week (reticulocyte count should improve within 1 week), resolution of neurologic symptoms may take much longer. Neurologic symptoms that persist for months or years become irreversible. In most elderly people with Vitamin B-12 deficiency and dementia, cognition does not improve after treatment. Table 6 lists the usual times until improvement for abnormalities associated with vitamin B12 deficiency ¹.

Vitamin B-12 treatment must be continued for life unless the pathophysiologic mechanism for the deficiency is corrected.

Infants of vegan mothers should receive supplemental Vitamin B-12 from birth.

In patients with a deficiency in intrinsic factor (IF), either due to pernicious anemia or gastric bypass surgery, a parenteral dose of vitamin B12 is recommended, as oral B12 will not be fully absorbed due to the lack of intrinsic factor. A dose of 1000 mcg of B12 via the intramuscular route is recommended once a month ¹⁵⁷. In newly diagnosed patients, 1000 mcg of vitamin B12 is given intramuscularly once a week for four weeks to replenish stores before switching to once-monthly dosing ¹⁵⁷. Studies have shown that at doses high enough to fully saturate intestinal B12 receptors, oral B12 is also effective, despite a lack of intrinsic factor ¹⁵⁷.

In anyone at risk of developing a B12 deficiency, such as patients with Crohn’s disease or celiac disease, routine monitoring of B12 should be performed. If the severity of the disease worsens and B12 levels begin to decline, treatment is then started. However, prophylactic treatment before B12 levels fall is not indicated ¹⁵⁹, ¹⁶⁰, ¹⁶¹.

Table 6. Usual times until improvement for abnormalities associated with vitamin B12 deficiency

[Source ⁵ ]

Vitamin B12 deficiency prognosis

For patients who are promptly treated with vitamin B12, the prognosis is good. In general, younger patients have better outcomes compared to older individuals. The best response is obtained in people with the absence of severe neurological deficits.

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